The broad long-term objectives of this proposal are to develop heat stable immunoassay reagents for Marburg and Ebola viruses and begin elucidating the molecular requirements for effective antigen capture assay performance. Both filoviruses continue to cause devastating outbreaks of hemorrhagic fever in Africa and threaten non-endemic countries, through the return of infected travelers, the importation of infected non-human primates and the threat of bioterrorism. The absence of vaccines and effective therapeutics means that rapid diagnosis is essential to contain an outbreak wherever it may occur. Current diagnostics are limited in terms of durability, portability and availability especially in resource poor settings and our aim is to augment these capabilities to help safeguard public health. We hypothesize that carefully chosen llama single domain antibody (sdAb) proteins specific for each of the five filovirus species will enable the assembly of rapid, accurate yet robust diagnostics. We base this hypothesis upon our studies that: 1. sdAbs selected to diverse antigens retain specific antigen binding activity when exposed to high temperatures unlike conventional immunoglobulins and 2. sdAbs selected to Marburg virus enable rapid antigen capture assays to be established that match nucleic acid based detection methods in terms of sensitivity and specificity. Our specific aims are to: 1. Characterize pre-existing sdAbs that have been selected against Marburg virus for their thermostability and requirements for molecular recognition;2. Characterize sdAbs selected on Ebola Zaire, Sudan, Ivory Coast and Reston to determine if the degree of assay sensitivity-specificity is conserved and if the sdAbs are thermostable;3. Begin to rationalize the molecular recognition of the Ebola sdAb and, in concert with the Marburg sdAb data, understand the rules and requirements for effective antigen capture assay development. These studies will guide the way to formulating inexpensive, simple yet ultrasensitive filoviral diagnostics with potentially infinite shelf lives even in the absence of refrigeration. PUBLIC HEALTH RELEVANCE: The filoviruses Marburg and Ebola continue to cause unpredictable outbreaks of transmissible and highly lethal hemorrhagic fevers in Africa. Other countries are also at risk through the importation of infected non-human primates, the return of infected tourists from endemic regions, and the threat of bioterrorism. Since no vaccines or therapeutics are currently available for these viruses, it is imperative to rapidly identify infected individuals on-site and quarantine them to reduce disease transmission. We aim to develop novel, durable antibodies specific for filoviral species to aid the development of inexpensive, rapid diagnostic tests to meet this urgent need. We also wish to study the mechanism of how these novel anti-filoviral antibodies can equal nucleic acid based detection limits to understand how to formulate improved virus antigen capture assays.